Genetic engineering techniques that provide for transferring a foreign, or exogenous, gene into a host's genome resulting in the production of a transgenic animal are among the most powerful tools available for the study of genetics and the understanding of genetic mechanisms. Although the field of transgenics was initially developed to understand the action of a single gene in the context of the whole animal and the phenomena of gene activation, expression, and interaction, this technology has shown great promise from an economic perspective. The use of transgenic technology to convert animals into “protein factories” for the production of specific proteins or other substances of pharmaceutical interest (Gordon et al., 1987, Biotechnology 5: 1183–1187; Wilmut et al., 1990, Theriogenology 33: 113–123) offers significant advantages over more conventional methods of protein production by gene expression. Likewise, the incorporation of an exogenous gene to produce an improved production animal could have important implications as, for example, in the production of a disease resistant bird.
Regulation signals, such as promoters and terminators, that allow ordered transcription are required to express foreign genes efficiently. Terminator sequences, located on the 3′ end of the encoding DNA, can serve to end transcription and, if appropriate, as a signal for polyadenylation of the mRNA formed. Promoter sequences, responsible for the expression of the foreign gene, contain recognition sequences for RNA-polymerases and for transcriptional effectors.
A large number of promoters suitable for controlling the expression of foreign genes axe known. For example, one of the most frequently used promoters, the cytomegalovirus immediate-early promoter, is described in U.S. Pat. No. 5,168,062 to Stinski. Because the CMV promoter provides for constitutive expression, a gene product under its regulation is expressed in most, if not all tissues.
Inducible or tissue-specific promoters may be employed to provide more selective gene expression. For example, U.S. Pat. No. 6,084,089 to Mine et al., discloses a promoter that induces gene expression at low temperatures in potato tubers, but which is scarcely induced at normal temperatures. Ryals et al., in U.S. Pat. No. 5,689,044, claim a chemically inducible promoter of a plant PR-1 gene, while a vector having a promoter that is inducible by methanol or glycerol is described in U.S. Pat. No. 5,750,372 to Sakai et al.
Examples of cell- and tissue-specific promoters include, inter alia, the following: a muscle-specific promoter associated with a avian retroviral vector described by Petropoulos, et al. (1992); a defective DNA viral vector having a neural tissue-specific promoter useful for in vivo expression of a gene (U.S. Pat. No. 6,040,172 to Kaplitt); an LPT2 promoter having aleurone-tissue specific activity (U.S. Pat. No. 5,525,716 to Olsen et al.); and promoters causing leaf-specific expression in plants, as disclosed by Sonnewald et al. in U.S. Pat. No. 6,229,067.
The failure of traditional methods such as vaccination and chemoprophylaxis in preventing avian infections associated with significant enteric pathogens such as Salmonella spp. makes producing disease resistant birds through transgenic technology an attractive option. A gene coding for an antimicrobial peptide and incorporated into a bird's genome could be capable of inhibiting the proliferation of a pathogen via specific or non-specific means. Novel peptides having antimicrobial activity, and DNA sequences encoding such peptides, include inter alia: purified bovine granulocyte peptide A and murine granulocyte peptide A (U.S. Pat. No. 6,008,195 to Selsted); antimicrobial peptides derived from lentiviruses (U.S. Pat. No. 5,945,507 to Montelaro et al.); DNA encoding biocidal proteins isolated from seeds which exhibit antifungal and antibacterial activity (U.S. Pat. No. 5,691,199 to Broekaert et al.); and an antimicrobial composition from a prokaryotic DNA extract (U.S. Pat. No. 6,096,719 to Matsutani et al.).
By placing the gene coding for an antimicrobial peptide under the control of a gut-specific promoter undesired side-effects associated with expressing the antimicrobial protein in a ubiquitous fashion can be minimized. In addition, a promoter capable of gut-specific expression would be useful when operably linked to other genes, especially those encoding proteins optimally localized to the gastrointestinal tract.
One means of identifying promoters exhibiting gut-specificity is by examining protein production in avian intestinal tissue. One such suitable candidate is intestinal fatty acid-binding protein (iFABP). The product of the FABP2 gene, iFABP is a member of a family of intracellular lipid-binding proteins and probably involved in the absorption and intracellular transport of dietary long-chain (C16–C20) fatty acids in the small intestine (Sacchettini et al., 1990; Schroeder et al., 1998; Hegele, 1998).
Members of the homologous, low molecular weight (15 kD), cytosolic lipid-binding proteins likely arose from an ancestor gene by repeated gene duplication, and include lipid-binding proteins specific to the liver, intestinal tissue, heart, ileal and adipocyte tissue, epidermis, brain, retinal tissue, retinoic acid, or peripheral myelin (Gordon et al., 1983; Alpers et al., 1984; Hayasaka et al., 1993; Shimizu et al., 1997; Schroeder et al., 1998). The tissue distributions of these gene products differ and are strictly regulated. Expression of iFABP, for example, is limited to the small intestinal epithelium, especially enterocytes and goblet cells, and not found in Paneth cells in the crypts or enteroendocrine cells (Sweetser et al., 1988a), even though all four types of cells originated from an identical stem cell (Schmidt et al., 1985).
Gene expression largely depends on the combination of the gene promoter sequence and transcription factors, and FABP promoters have provided one of the best models for studying tissue-specific gene regulation and cell differentiation in vertebrates. A cis-acting promoter sequence for iFABP has been characterized in mammals (rat, mouse, and human) and amphibia (Xenopus) (Sweetser et al., 1987; Green et al., 1991; Gao et al. 1998). The sequences located between nucleotides −277 and +28 (or more concisely −103 and +28) from the transcription start site appear to be important for directing gut- and cell type-specific expression of the rat iFABP gene (Sweetser 1988a, b; Rottman and Gordon, 1993). That this region of the rat iFABP promoter can direct tissue-specific gene expression in a transgenic frog suggests conservation of the regulatory mechanism of iFABP expression among vertebrates (Beck and Slack, 1999). Several elements in the proximal 0.3 kb region have been nominated as regulatory sequences involved in tissue specific expression of iFABP, particularly in the rat. For example, a 14-bp element composed of two direct 7-bp repeats is conserved among the gene promoters of several small intestine-specific genes in mammals. Two members of the steroid hormone receptor superfamily, HNF-4 and ARP-1 are reported to bind to the iFABP promoter element (Issemann and Green, 1990; Rottman and Gordon, 1993).
The amphibian iFABP gene promoter lacks a peroxisome proliferator-responsive element (PPRE)-homologous element, and the importance of this element and transcriptional factors in the tissue-specific expression of iFABP, therefore, is obscure. It has been suggested that binding of GATA-4 and -5 to a proximal GATA-binding site is involved in tissue specific expression of the iFABP gene in vitro (Gao et al., 1998). However, gene activation by these transcription factors is modest, and additional cell-specific factors are probably required for in vivo regulation.
A 20-bp cis-acting element that regulates cell lineage-specific patterns of iFABP expression has been identified by promoter mapping studies in transgenic mice (Simon et al., 1995). This element, located from −263 to −244 in the rat FABP2 gene, binds small intestinal nuclear proteins and acts as a suppressor of gene expression in iFABP-negative intestinal epithelial cells such as colon epithelium, and cells located in the crypts of Lieberkuhn, and the Paneth cell lineage. The short (−277 to +28 bp) promoter of rat iiFABP showed rather weak promoter activity when compared to the long (1.2 kb) promoter in the small intestine and failed to direct expression of the gene in the ileum (Sweetser et al., 1988a). Thus, the proximal 0.3 kb promoter region is very important in the regulation of iFABP gene, but the more distal sequence also contributes to precise control of this gene.